Nowadays, many efforts are being made in order to obtain less expensive and renewable sources of fuel. Biofuels offer an attractive alternative to petroleum based fuels and can be obtained through the fermentation of monomeric sugars derived from starch or cellulose. However, current economics do not support the widespread use of biofuels due to the high cost of generating them.
Plant biomass provides a plentiful source of potential energy in form of sugars that can be utilized for numerous industrial and agricultural processes, and is therefore a significant renewable resource for the generation of fermentable sugars that can yield commercially valuable end-products, such as biofuel. However, the enormous energy potential of these carbohydrates is currently under-utilized because the sugars are locked in complex polymers and, hence, are not readily accessible for fermentation (WO2012018691A2).
Wood, agricultural residues, herbaceous crops, and municipal solid wastes have been considered as feedstocks for ethanol production. These materials primarily consist of cellulose, hemicellulose, and/or lignin. Once the cellulose is converted to glucose by means of an enzymatic hydrolytic process, the glucose is easily fermented by yeast into ethanol. Thus, the more amounts of complex sugars remaining at the end of the hydrolytic process the lower the yield of ethanol production at the end of the fermentation process. Therefore, one area of research aimed at decreasing costs and enhancing the yield of biofuel production processes is focus on the enhancement of the technical efficacy of the hydrolytic enzymes that can be used to generate fermentable sugars from biomass.
Due to the complexity of biomass, its conversion to monomer sugars involves the action of several different enzyme classes, which digest cellulose and hemicellulose, major polysaccharides comprised in cellulosic materials. After cellulose, hemicellulose is the second most abundant fraction available in nature. It is a storage polymer in seeds and it forms the structural component in cell walls of woody plants. The classification of these hemicellulose fractions depends on the types of sugar moieties present. The principal monomers present in most of the hemicelluloses are D-xylose, D-mannose, D-galactose and L-arabinose. Thus, hemicellulose includes xylan, mannan, galactan and arabinan as the main heteropolymers. Specifically, xylan contains 85 to 93% of D-xylose, a small amount of c-arabinose and traces of glucuronic acid residues. The main chain of xylan is composed of β-(1-4) linked β-xylopyranose residues, and several side chains have been described to be present. Among them, most usually found are xylopiranose, glucuronic acid and arabinofuranose linkages, as well as acetyl groups (Bastawde, 1992, World Journal of Microbiology and Biotechnology (8) 353-368).
The presence of lignin in biomass leads to a protective barrier that prevents proper enzymatic hydrolysis of glucan and xylan. Thus, a pretreatment process of the biomass is required for increasing the access of the enzymes to their substrates and consequent efficient hydrolysis. Pretreatment uses various techniques, including ammonia fiber explosion, chemical treatment and steam explosion at high temperatures to alter the structure of cellulosic biomass and make cellulose more accessible. Hemicellulose can be readily hydrolysed under moderate conditions, but much more extreme conditions are needed for cellulose hydrolysis. Therefore, the pretreated material (substrate for the enzymatic hydrolysis) usually contains a high concentration of xylose, whereas glucose content is rather low (Kumar et al, 2009. Ind. Eng. Chem. Res., 48 (8), 3713-3729).
Single component enzymes have been shown to only partially digest cellulose and hemicellulose and thus the concerted action of different classes of enzymes is required to complete their conversion to monomeric sugars. Many more enzymes are required to digest hemicellulose to sugar monomers including xylanase, xylosidase, arabinofuranosidase, mannanase, galactosidase and glucuronidase. Non-glycosyl hydrolases such as acetyl xylan esterase and ferulic acid esterase may also be involved.
A large number of naturally-occurring organisms have been found to produce enzymatic hydrolysis of cellulosic materials to produce fermentable sugars. Organisms capable of carry out a complete cellulose and hemicellulose degradation, that subsequently allows an efficient fermentation, would greatly enhance the cost effectiveness of bioethanol production.
The hydrolytic efficiency of a multi-enzyme complex in the process of cellulosic saccharification (or hydrolysis) depends both on properties of the individual enzymes and the ratio of each enzyme within the complex. It is therefore desirable to generate cellulolytic enzymes expressing-microorganisms which improve the yield of cellulosic material degradation process, increasing the amount of released fermentable sugars and thus improving the yield of final biofuel production.
Thus, some efforts carried out in order to generate improved cellulolytic enzymes expressing-microorganisms have involved inserting a gene encoding the specific hydrolytic enzyme to be expressed under the control of strong expression signals, which leads to an increased stability of the transcribed mRNA or an increased number of copies of the gene in the produced organism (US20080194005A1).
A number of host cells used for heterologous gene expression, such as bacteria Escherichia coli, and methods of transformation have been disclosed in the prior art. In this context, also a number of fungal expression systems have been developed, for instance Aspergillus niger, Aspergillus awamori, Aspergillus nidulans, Trichoderma reesei. However, for various reasons many of these recombinant microorganisms have not found widespread acceptance or use. In general terms, the ideal host cell must fulfill a large number of criteria, such as, uses the medium efficiently, produces the polypeptide or protein of interest in high yield, should be capable of efficient secretion of the protein or polypeptide, allows a wide range of expression regulatory elements to be used thus ensuring ease of application and versatility, allows the use of easily selectable markers that are cheap to use, and produce stable transformants.